NBT/BCIP in situ detection problem

[Peter Ashby]

In article <39F016F8.455732A7 at neurochem.u-strasbg.fr>, Paul Klosen 
<klosen at neurochem.u-strasbg.fr> wrote:

> Hi,
> 
> Recently we have run into a nasty problem with our NBT/BCIP in situ
> detections. The in situ part seems to work fine and the alk phos
> detections is sensitive, but when we observe our slides under the
> microscope, the precipitate is completely granular and seems to be
> located above the tissue. We have tried varying several parameter (pH,
> salt and MgCl2 conc., ..) and different lots and sources of both NBT and
> BCIP, but nothing seems to bring us back to our previous results, where
> we had a nice cell-filling precipitate, albeit a bit diffuse as expected
> with NBT/BCIP.
> 
> Has anyone encountered a similar problem, and how did you get rid of it
> ?
> Thanx

Are you sure it is a problem with the in situ rather than your 
microscopy which could produce these symptoms? Are you doing in situs on 
sections or are you sectioning whole mounts? Either way it could 
indicate that your staining is simply stronger than it used to be, you 
could try incubation for less time.

Peter

-- 
Peter Ashby
Wellcome Trust Biocentre
University of Dundee
Dundee, Scotland
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